Glutathione (GSH) is a compound which, in its reduced form, constitutes an excellent antioxidant and therefore a defence against the damage caused by free radicals to higher organisms. S-acetyl-glutathione (SAG) (see FIG. 1) is a synthetic derivative thereof which protects GSH against oxidation, and simultaneously releases it easily by hydrolysis.
GSH or γ-L-glutamyl-L-cysteinylglycine is a tripeptide consisting of glutamic acid, cysteine and glycine, characterised by an atypical peptide bond, namely the bond that binds the nitrogen of cysteine to the carboxyl in γ glutamic acid. It is the main thiol compound with the lowest molecular weight present in both animal and plant cells (about 95% of the total). Its function is to maintain in the reduced state the —SH groups of many enzymes and proteins whose oxidation (with the formation of S—S intra- and intermolecular disulphide bridges) leads, in most cases, to inactivation or loss of the biological function of the enzyme or protein.
GSH is considered to be one of the most important intracellular antioxidants produced naturally by the human body. However, chronic oxidative stress reduces the cell levels of GSH, and it is often appropriate to replenish its levels with the aid of diet supplements.
It is commonly believed that the GSH intake obtained from the diet or with the use of diet supplements is easily used by the issues, but in reality it is not absorbed “as is”, but hydrolysed into its three constituent amino acids by a gamma-glutamyl transpeptidase present in the intestine. After being absorbed and introduced into the bloodstream, said amino acids are distributed to the various tissues wherein they implement the pool of amino acids with which the body cells synthesise endogenous GSH. It is therefore necessary to use a high oral dose in order to guarantee significant absorption. When Witschi et al. evaluated the increase in the blood levels of glutathione, cysteine and glutamate after oral administration of GSH to seven healthy volunteers, no significant increases were observed at doses of up to 3 g per dose (Witschi A et al., J. Clin. Pharmacol. 43 (6), 667-1992).
Sublingual administration, which guarantees better bioavailability, can be used as an alternative to oral administration of GSH.
Finally, in the pharmaceutical field, prophylaxis based on GSH is used in some cases by parenteral, intramuscular or slow intravenous administration, for example as prophylaxis for neuropathy resulting from chemotherapy with cisplatin or analogues.
The use of SAG as a precursor is a good alternative to replenish the reduced GSH levels in the body. In fact, acetylation of the sulphur atom prevents the decomposition of GSH and facilitates its absorption through the intestinal wall, thus enabling the molecule to pass extensively into the cells.
The SAG thus assimilated by the tissues is hydrolysed by cytoplasmic thioesterase and, by hydrolysis of the acetyl group, produces reduced GSH which is available for all the biological functions wherein it is required.
The addition of SAG to cultures of fibroblasts originating from individuals suffering from a genetic glutathione synthetase deficiency has proved able to replenish the intracellular level of GSH effectively (Okun J G et al., J. Inherit. Metab. Dis. 27(6), 783-2004). SAG is also more stable in the plasma and has proved more effective than GSH in replenishing the cell levels of GSH impoverished by viral infections (Vogel J U et al., Med. Microbiol. Immunol. 194, 55-2005) (Fraternale A et al., Antiviral Res. 77, 120-2008). Finally, SAG exhibits an interesting non-GSH-dependent activity that induces apoptosis in some human tumour cell lines in vitro. (Locigno R et al., Int. J. Oncol. 20, 69-2002).
Identification and characterisation of the polymorphic forms, and of the experimental conditions for obtaining them, are very important parameters for a compound designed for nutraceutical and/or pharmaceutical use.
The synthesis of SAG has already been claimed in a Japanese patent (see Chemical Abstract 97-7222755s) and in WO92/00320. However, the authors only disclose a general method for obtaining it, without investigating the existence of polymorphic forms in any way.
As stated above, a number of difficulties are involved in the absorption of GSH, which are partly solved by the use of the SAG derivative. However, the absorption of said compound may be adversely affected by the existence of polymorphic forms thereof having different physicochemical characteristics that influence its dissolution rate, solubility and therefore bioavailability, not to mention the different behaviour of the powders during the preparation of the various formulations.
No experimental condition or preliminary indication for crystallisation and drying that suggests the existence of polymorphic forms of SAG has ever been disclosed in any patent or patent application.